The long term goal of this application is to learn how the amounts and release of the endogenous opioid peptides are regulated. This information is critical for an understanding of the role of these peptides in opioid tolerance and dependence and in discovering low to use the endogenous opioid peptides as a new method for the management of pain. The objectives of this proposal are to describe and define the factors that regulate opioid peptide gene expression, biosynthesis and release and to determine whether treatments that may alter opioid peptides and their mRNAs are linked to the function (e.g., analgesia) of the endogenous opioid peptides. In addition to a quantitative Northern blot analysis procedure already in use, methods will be developed or adapted for the quantitation of opioid peptide mRNAs, their processing and localization including a new quantitative solution by hybridization assay, a nuclear run-on transcription assay, a primary transcript protection assay and an in situ hybridization technique. Opioid peptide gene expression and the relative amounts of opioid peptide precursors and posttranslational processing products will be measured by use of radioimmunoassays, size exclusion and high performance liquid chromatographic methods we have developed. The targets for study include the CNS (striatum, hippocampus, hypo- thalamus and pituitary) and the adrenal medulla of rats and hamsters at different postnatal and adult ages, during and after acute and chronic opioid agonist and antagonist treatment and following opioid and nonopioid stressors. These later studies will attempt to link biochemical changes in the levels and the release of the endogenous opioid peptide system to functional effects such as analgesia. Explants of rat adrenal medulla and striatum and a human glioma cell line will be used to examine the co-regulation of opioid peptides and co-localized catecholamine and to define the mechanisms of hormonal and neurogenic regulation of opioid peptide gene expression and biosynthesis.